AAA+ proteases remove toxic proteins from cells and regulate many other important cellular processes that are required to promote health and prevent disease. As protein degradation is irreversible, it must be carefully regulated. The architectures of AAA+ proteases and the principles of degradation control are similar in all organisms. AAA+ proteases assemble into multi-subunit structures with an internal proteolytic chamber, accessible through narrow channels that exclude natively folded proteins. This mechanism protects most proteins from unintended degradation and requires specific substrates to be recognized, unfolded, and then translocated into the degradation chamber. In the AAA+ ClpXP protease, for example, a ring hexamer of ClpX uses the energy of ATP hydrolysis to unfold specific target proteins and translocates them into ClpP for degradation. ClpXP is one of the best-characterized AAA+ proteases and is a paradigm for other ATP-dependent proteases and AAA+ remodeling machines. These ATP-fueled enzymes perform a wide variety of remodeling, transport, and regulatory tasks in the cell, all of which require mechanical work. In mammals, loss of mitochondrial ClpP results in infertility, hearing loss, and a growth defect, whereas mitochondrial ClpX plays an important role in heme biosynthesis. Bacterial ClpXP can promote pathogenesis and is a validated antibiotic target in M. tuberculosis. Substantial progress has been made in understanding the general biochemical and structural features of ClpXP and other AAA+ enzymes but important and fundamental questions concerning the molecular mechanisms of these machines remain unanswered. For example, it is not known how ClpX grips and interacts with polypeptide substrates during mechanical unfolding and translocation, how the hexameric ClpX ring engages protein substrates and coordinates ATP binding and hydrolysis with conformational switching between nucleotide loadable and unloadable subunits during function, or how ClpX binds and collaborates with ClpP during degradation. The experiments described in this proposal will answer these questions and provide a conceptual framework and a set of novel tools applicable to studies of the entire superfamily of AAA+ machines.